UNIVERSITÀ DEGLI STUDI DI SASSARI
Scuola di Dottorato di Ricerca in
Scienze Biomolecolari e Biotecnologiche
Indirizzo: Biochimica e Biologia Molecolare
XXIV ciclo
Study of intracellular signaling pathways triggered
by natural antioxidants in human endothelial cells
Coordinatore:
Prof. Bruno Masala
Tutor:
Prof. Gianfranco Pintus
Tesi di Dottorato di:
Dott.ssa Annalisa Cossu
Anno Accademico 2010-2011
Index
INDEX
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
1
Index
CHAPTER 1 INTRODUCTION
pag 4
§ 1.1 Chemistry of ROS
pag. 5
§ 1.2 Sources of ROS
pag. 7
§ 1.3 Role of ROS
pag. 13
§ 1.4 Antioxidants
pag. 14
§ 1.5 ROS and cell signalling
pag. 21
§ 1.6 ROS and apoptosis
pag.28
CHAPTER 2 AIM OF THE WORK
pag. 32
CHAPTER 3 MATERIALS AND METHODS
pag. 36
§ 3.1. Reagents
pag. 37
§ 3.2 Cell culture and treatments
pag. 37
§ 3.3 Measurements of intracellular ROS
pag. 38
§ 3.4 Measurements of protein carbonylation
pag. 38
§ 3.5 Measurement of NADH and NADPH consumption
pag. 39
§ 3.6 Cell viability assay
pag. 40
§ 3.7 Cell metabolic assay
pag. 41
§ 3.8 Cell apoptosis assay
pag. 42
§ 3.9 Immunoblotting analysis
pag. 43
§ 3.10 MMP assay
pag. 43
§ 3.11 Statistical analysis
pag. 45
CHAPTER 4 RESULTS
pag. 46
§ 4.1 Dose-dependent effect of NA on EC ROS levels and
protein carbonylation
§ 4.2 NA dose dependently induce ECs impairment
pag. 47
pag. 50
§ 4.3 Flavin-containing oxidases mediate NA-induced intracellular
ROS production and protein carbonylation
§ 4.4 NA dose dependently downregulate Akt phosphorylation
pag. 55
pag. 57
§ 4.5 Akt dephosphorylation and EC damage are mediated by
flavin oxidases
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
pag. 60
2
Index
§ 4.6 Akt activation rescues ECs from oxidative stress damage
pag. 62
§ 4.7 CYP2C9 mediate NA-induced ECs damage
pag. 66
§ 4.8 SPZ prevents NA-induced p-Akt down-regulation
pag. 69
§ 4.9 Cyclosporine A prevents NA-induced ECs impairment
pag. 70
CHAPTER 5 DISCUSSION
pag. 72
CHAPTER 6 BIBLIOGRAFY
pag. 81
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
3
Introduction
CHAPTER 1
INTRODUCTION
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
4
Introduction
Reactive Oxygen Species (ROS)
§ 1.1 Chemistry of ROS
Reactive Oxygen Species (ROS) are a variety of molecules and free
radicals derived from molecular oxygen.
A radical is a clusters of atoms one of which contains an unpaired electron in
its outermost shell of electrons. This is an extremely unstable configuration,
and radicals quickly react with other molecules or radicals to achieve the
stable configuration of 4 pairs of electrons in their outermost shell (one pair
for hydrogen).
Molecular oxygen in the ground state is a bi-radical, containing two unpaired
electrons in the outer shell (also known as a triplet state). Since the two single
electrons have the same spin, oxygen can only react with one electron at a
time and therefore it is not very reactive with the two electrons in a chemical
bond. On the other hand, if one of the two unpaired electrons is excited and
changes its spin, the resulting species (known as singlet oxygen) becomes a
powerful oxidant as the two electrons with opposing spins can quickly react
with other pairs of electrons, especially double bonds.
Oxygen is a crucial molecule to higher organisms life, acting as final acceptor
for the electron released during biologic oxidations, under aerobic conditions.
The superior role of aerobic versus anaerobic metabolism depends on the
higher energy yeld; so the aerobic respiration is the normal procedure for
energy production in animal cells. Only a few mammalian tissues (notably
erythrocytes and skeletal muscle) have developed major ability to obtain
energy under anaerobic conditions from carbohydrate metabolism, through
the glycolytic pathway.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
5
Introduction
The reduction-oxidation reactions occur during normal metabolic processes:
during normal respiration, molecular oxygen is sequentially reduced by the
addition of four electrons to generate water.
The reduction of oxygen by one electron at a time produces several different
oxygen metabolites, which are collettively called ROS.
Superoxide anion (O2−•), the product of a one-electron reduction of oxygen, is
the precursor of most ROS and a mediator in oxidative chain reactions.
Dismutation of O2−• (either spontaneously or through a reaction catalysed by
superoxide dismutases) produces hydrogen peroxide (H2O2), which in turn
may be fully reduced to water or partially reduced to hydroxyl radical (•OH),
one of the strongest oxidants in nature. The formation of •OH is catalysed by
reduced transition metals, which in turn may be re-reduced by O2−•,
propagating this process [1]. In addition, O2−• may react with other radicals
including nitric oxide (NO•) in a reaction controlled by the rate of diffusion of
both radicals. The product, peroxynitrite (ONOO−), is also a very powerful
oxidant [2]. The oxidants derived from NO• have been recently called
reactive nitrogen species (RNS).
The structure of ROS is shown in the figure below, along with the notation
used to denote them.
Another radical derived from oxygen is singlet oxygen, designated as 1O2•.
This is an excited form of oxygen in which one of the electrons jumps to a
superior orbital following absorption of energy.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
6
Introduction
§ 1.2 Sources of ROS
As mentioned above, the reactive oxygen species are the main
byproducts formed in the cells of aerobic organisms, and can initiate
autocatalytic reactions so that molecules to which they react are themselves
converted into free radicals to propagate the chain of damage.
The amount of free radical production is determined by the balance of many
factors, and ROS are produced both endogenously and exogenously.
In mammalian cells the endogenous sources of ROS include:
 mitochondria (mainly complex I & III, but also monoamino oxidase, αketoglutarate dehydrogenase, glycerol phosphate dehydrogenase, p66shc
[3]);
 endoplasmic reticulum (mainly cytochrome P-450 and b5 enzymes,
diamine oxidase, Ero1 [4]);
 peroxisomes (mainly fatty acid oxidation, D-amino acid oxidase, L-2hydroxyacid oxidase and urate oxidase [5]);
 cytosol (NO synthases, lipoxygenases and PGH synthase [6 - 8])
 plasma membrane (NADPH oxidases, lipoxygenase [9; 10]) and
extracellular space (xanthine oxidase [11]).
Mitochondria seem to be (quantitatively) the most important subcellular site
of O2−• and H2O2 production in mammalian organs and the steady state
concentration of O2−• in the mitochondrial matrix is about 5- to 10-fold higher
than that in the cytosolic and nuclear spaces [12].
Approximately 98% of the oxygen we metabolize undergoes a concerted
tetravalent reduction to produce water in a reaction catalyzed by cytochrome
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
7
Introduction
oxidase (cytochrome c-oxygen oxidoreductase) of complex IV in the
mitochondrial electron transport chain (O2 + 4e− + 4H+
2H2O).
The Electron Transport Chain (ETC) is an essential mechanism for generation
of cellular energy and it is localized to the mitochondrial inner membrane.
The ETC consists of four multi-subunit enzyme complexes and the mobile
electron carriers, coenzyme Q (inner membrane) and cytochrome c
(intermembrane space), that pass electrons sequentially from high (NADH or
FADH2) to low (molecular oxygen) redox potential.
Cytochrome oxidase is the terminal electron acceptor in the chain and must
give up its reducing equivalents to allow continued electron transport: if
electrons stop flowing through the chain, the protonmotive force dissipates
and ATP production cannot continue.
During normal respiration, approximately 2–4% of electron flow through the
ETC results in only partial reduction of O2, generating superoxide (O2−•)
which dismutates to form hydrogen peroxide (H2O2), which can further react
to form the hydroxyl radical (HO•).
Although the mitochondrial electron transport chain is a very efficient system,
the very nature of the alternating one-electron oxidation-reduction reactions it
catalyzes (generating a constantly alternating series of “caged” radicals),
predispose each electron carrier to side reactions with molecular oxygen.
Thus, for example, as ubiquinone within the electron transport chain cycles
between the quinone (fully oxidized) to semiquinone (one-electron reduction
product) to quinol (fully reduced by two electrons) states, there is a tendency
for an electron to pass to oxygen directly (generating O2−•) instead of to the
next electron carrier in the chain.
There are two specific sites where electrons may leak out of the chain to
partially reduce oxygen. One is the NADH dehydrogenase in complex I and
the other is the ubiquinone–cytochrome c reductase in complex III [13; 14]. In
contrast, although complex IV contains intermediates corresponding to the
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
8
Introduction
three oxyradicals, O2−•, H2O2 and •OH, these intermediates are tightly bound
and chemically „„disguised‟‟, preventing production of reactive oxygen
species by complex IV [15].
Several iron-sulfur clusters within the respiratory chain are also subject to
such toxic, O2−• generating, side reactions with oxygen. Thus it is commonly
held that mitochondrial generation of O2−• represents the major intracellular
source of oxygen radicals under physiological conditions.
In addition to these toxic electron transport chain reactions of the inner
mitochondrial membrane, also the mitochondrial outer membrane enzyme,
monoamine oxidase, is a quantitatively large source of H2O2 that contributes
to an increase in the steady state concentrations of reactive species within
both the mitochondrial matrix and cytosol [12]. Monoamine oxidase is a
flavoprotein ubiquitously expressed in higher eukaryotic organisms and it
catalyzes the oxidative deamination of primary aromatic amines along with
long-chain diamines and tertiary cyclic amines. The enzyme-catalyzed
oxidative deamination of biogenic amines is a quantitatively large source of
H2O2 that contributes to an increase in the steady state concentrations of
reactive species within both the mitochondrial matrix and cytosol.
Reactive oxygen species cause damage to mitochondrial components
and initiate degradative processes. The sites of „„electron leak‟‟ during normal
respiration provide insight into the likely sources of increased oxyradical
production in pathologic states.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
9
Introduction
Reactive oxygen species generation and disposal in the mitochondria (from: J. Cell Biol.; 2011; Vol. 194 No.
1 7–15. T. Finkel: Signal transduction by reactive oxygen species.)
The endoplasmic reticulum (ER) is a membrane-bound intracellular
organelle that is primarily involved in lipid and protein biosynthesis. Smooth
ER (lacking bound ribosomes) contains enzymes that catalyze a series of
reactions to detoxify lipid-soluble drugs and other harmful metabolic
products. The most extensively studied of these are the cytochrome P-450.
Cytochrome P450s comprise a superfamily of heme-thiolate proteins
named for the spectral absorbance peak of their carbon-monoxide-bound
species at 450 nm. They support the oxidative, peroxidative and reductive
metabolism of such endogenous and xenobiotic substrates as environmental
pollutants, agrochemicals, plant allelochemicals, steroids, prostaglandins and
fatty acids .
Cytochrome P450s have traditionally been referred to as hydroxylases, mixed
function oxidases and monooxygenases [16]. Their main function is to
activate molecular oxygen to yield a reactive species that can attack relatively
inert chemical sites in order to introduce hydroxyl groups into structures
unreactive as hydrocarbon chains and aromatic rings. This serves to facilitate
the biotransformation of compounds that would otherwise lack functional
groups suitable for conjugation. In addition to hydroxylations, however,
cytochrome P450s also catalyze a broad variety of other chemical reactions
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
10
Introduction
including deaminations, desulfurations, dehalogenations, epoxidations, N-, S-,
and O-dealkylations, N-oxidations, peroxidations and sulfoxidations.
Cytochrome P450 has also been proposed as a source of reactive oxygen
species. Through the induction of cytochrome P450 enzymes, the possibility
for the production of reactive oxygen species, in particular, superoxide anion
and hydrogen peroxide, emerges following the breakdown or uncoupling of
the P450 catalytic cycle [17]. In fact the underlying concept of its activity is a
multi step transfer of 2 electrons to a substrate while binding one oxygen
atom to it, the second being reduced to water. Part of the oxygen involved is
inevitably reduced to superoxide.
The catalytic cycle of cytochrome P450 (from: J. Phys. Chem. B; 2007, 111, 4251-4260Y. Wang, Yan Li and
B. Wang: Stochastic Simulations of the Cytochrome P450 Catalytic Cycle.).
Also peroxisomes are an important source of cellular H2O2 production
[5]. They contain a number of H2O2-generating enzymes including glycolate
oxidase, D-amino acid oxidase, urate oxidase, L--hydroxyacid oxidase and
fatty acyl-CoA oxidase. Peroxisomal catalase utilizes H2O2 produced by these
enzymes to oxidize a variety of other substrates in “peroxidative” reactions
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
11
Introduction
[18]. These types of oxidative reactions are particularly important in liver and
kidney cells in which peroxisomes detoxify a variety of toxic molecules
(including ethanol) that enter the circulation. Another major function of the
oxidative reactions carried out in peroxisomes is -oxidation of fatty acids,
which in mammalian cells occurs in mitochondria and peroxisomes [19].
Specific signaling roles have not been ascribed to peroxisome-derived
oxidants, and only a small fraction of H2O2 generated in these intracellular
organelles appears to escape peroxisomal catalase [18].
In the cytosol, the arachadonic acid cascade, yielding prostaglandins
and leukotrienes may generate ROS when the released lipid is metabolized.
ROS can be generated as byproducts during metabolism of arachidonic acid,
which to some degree takes place in practically every cell. Enzymes
participating in the process are cyclooxygenase, lipooxygenase and
cytochrome P-450 [20]. Arachidonic acid may be a source of ROS even by a
non-enzymatic process.
Instead the best characterized of the plasma membrane oxidases in
general is the NADPH oxidase. It is present in both professional phagocytic
cells (macrophages, neutrophils and eosinophils) and nonphagocytic cells
(such as endothelia or smooth muscle cells) [21]. This multicomponent
enzyme catalyzes the one-electron reduction of O2 to O2−•, with NADPH as
the electron donor through the transmembrane protein cytochrome b558 (a
heterodimeric complex of gp91phox and p22phox protein subunits). The transfer
of electrons occurs from NADPH on the inner aspect of the plasma membrane
to O2 on the outside.
During phagocytosis, the plasma membrane is internalized as the wall of the
phagocytic vesicle, with what was once the outer membrane surface now
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
12
Introduction
facing the interior of the vesicle. This targets the delivery of O2−• and its
reactive metabolites internally for localized microbicidal activity [22].
The NADPH oxidase of nonphagocytic cells is similar in structure and
it is activated by various hormones and cytokines. It is permanently in a fully
preassembled state and constantly producing low amounts of O2−•, most likely
with a regulatory function [23].
NADPH oxidase superoxide production is directed mainly intracellularly as
opposed to NADPH oxidase generated superoxide in neutrophils, where it
serves as a defence mechanism outside the cell.
In addition to physiological sources of ROS, diverse exogenous agents
can contribute to the intracellular production of free radicals. Most of these
compounds cause the generation of superoxide (O2−•) and hydrogen peroxide
(H2O2). The mechanism of action of many exogenous agents involves redox
cycling whereby an electron is accepted to form a free radical and it is then
transferred to oxygen. The exogenous sources of ROS are: xenobiotics,
chlorinated compounds, environmental agents, metals (redox and nonredox),
ions, and radiation [1].
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
13
Introduction
The sources and cellular responses to ROS (from: Nature; Nov 9 2000; 408(6809): 239-47T. Finkel & N. J.
Holbrook: Oxidants, oxidative stress and the biology of ageing).
§ 1.3 Role of ROS
It has been established that ROS can be both harmful and beneficial in
biological systems depending on the environment [24; 25]. Beneficial effects
of ROS involve, for example, the physiological roles in cellular responses to
noxia such as defense against infectious agents, and in the function of a
number of cellular signaling systems. In contrast overproduction of free
radicals can cause oxidative damage to biomolecules (lipids, proteins, DNA),
eventually leading to many chronic diseases such as atherosclerosis, cancer,
diabetics, rheumatoid arthritis, post-ischemic perfusion injury, myocardial
infarction, cardiovascular diseases, chronic inflammation, stroke and septic
shock, aging and other degenerative diseases in humans [26]. It has been
reported that deleterious effects of ROS on human cells may end in oxidative
injury leading to programmed cell death i.e. apoptosis [27].
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
14
Introduction
§ 1.4 Antioxidants
Humans have evolved highly complex antioxidant systems (enzymatic
and nonenzymatic), which work synergistically, and in combination with each
other to protect the cells and organ systems of the body against free radical
damage. The term “antioxidant” refers to any molecule capable of stabilizing
or deactivating free radicals before they attack cells. An ideal antioxidant
should be readily absorbed and quench free radicals, and chelate redox metals
at physiologically relevant levels.
Excessive production of ROS, outstripping endogenous antioxidant defense
mechanisms, determines the degree of oxidative stress.
The most efficient enzymatic antioxidants involve superoxide dismutase
(SOD), catalase and glutathione peroxidase [22]. SOD speeds the conversion
of superoxide to hydrogen peroxide, whereas catalase and glutathione
peroxidase convert hydrogen peroxide to water.
First in the line of enzymatic ROS degradation is superoxide dismutase
(SOD). This enzyme exists in 3 forms: a) Cu/Zn SOD present mainly in
cytosolic matrix, b) MnSOD localized preferentially in mitochondria and c)
extracellular SOD.
The SODs catalyze the reaction: O2−• + O2−• + 2H+
H2 O2 + O2
This dismutation, or disproportionation reaction, makes use of the fact that
superoxide is both an oxidant and a reductant, eager to get rid of its extra
electron or to take on another. The enzyme uses one superoxide radical to
oxidize another.
Catalase works in much the same way, because hydrogen peroxide can
be a weak reductant as well as a fairly strong oxidant; it catalyzes the
reaction: H2O2 + H2O2
2H2O + O2
This enzyme is present in the peroxisome of aerobic cells and it is very
efficient in promoting the conversion of hydrogen peroxide to water and
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
15
Introduction
molecular oxygen. Catalase has one of the highest turnover rates for all
enzymes: one molecule of catalase can convert approximately 6 million
molecules of hydrogen peroxide to water and oxygen each minute [1].
Glutathione peroxidase acts in association with tripeptide glutathione
(GSH), which is present in high concentrations in cells and catalyzes the
conversion of hydrogen peroxide or organic peroxide to water or alcohol
while simultaneously oxidizing GSH. It also competes with catalase for
hydrogen peroxide as a substrate and it is the major source of protection
against low levels of oxidative stress [1]. This enzyme uses NADPH as the
reducing species for hydrogen peroxide:
NADPH + H+ + H2O2
2H2O + NADP+.
Glutathione peroxidase can reduce lipid peroxides as well as hydrogen
peroxide and it is a very important enzyme in the prevention of lipid
peroxidation to maintain the structure and function of biologic membranes.
The non-enzymatic antioxidants are mostly “scavengers” of free
radicals, such as vitamin C, vitamin E (inhibits oxidation of membrane
lipids), uric acid (efficient scavenger of peroxynitrite, present in plasma and
airway lining fluid), albumin, bilirubin, glutathione or N-acetylcysteine
(NAC). NAC is a potent drug which acts directly by reacting with ROS
(forming NAC disulfides in the end) and indirectly, serving as a GSH
precursor [28]. However, as with many antioxidant substances, NAC in high
doses can exhibit prooxidative effects [29].
Although the metal chelating plasma proteins (transferrin, ceruloplasmin,
albumin, haptoglobin, and hemopexin) do not interact directly with and
decompose ROS, they are considered antioxidants because they bind redox
active metals and limit the production of metal-catalyzed free radicals. So the
maintenance of intracellular redox homeostasis is dependent on a complex
web of antioxidant molecules.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
16
Introduction
The antioxidants can be endogenous or obtained exogenously, eg as a
part of a diet or as dietary supplements. Some dietary compounds that do not
neutralize free radicals, but enhance endogenous activity may also be
classified as antioxidants.
Endogenous antioxidants play a crucial role in maintaining optimal cellular
functions and thus systemic health and well-being. However, under conditions
which promote oxidative stress, endogenous antioxidants may not be
sufficient and dietary antioxidants may be required to maintain optimal
cellular functions.
Polyphenols are the most abundant antioxidants in our diet and are
widespread constituents of fruits, vegetables, cereals, olive, dry legumes,
chocolate and beverages, such as tea, coffee and wine [30]. Despite their wide
distribution, the healthy effects of dietary polyphenols have come to the
attention of nutritionists only in the last years. The main factor responsible for
the delayed research on polyphenols is the variety and the complexity of their
chemical structure. Polyphenols comprise a wide variety of molecules that
have a polyphenol structure (i.e. several hydroxyl groups on aromatic rings),
but also molecules with one phenol ring, such as phenolic acids and phenolic
alcohols. Polyphenols are divided into several classes according to the
number of phenol rings that they contain and to the structural elements that
bind these rings to one another.
The main groups of polyphenols are: flavonoids, phenolic acids, phenolic
alcohols, stilbenes and lignans. The flavonoids, which share a common
structure consisting of 2 aromatic rings (A and B) that are bound together by 3
carbon atoms that form an oxygenated heterocycle (ring C), may themselves
be divided into 6 subclasses as a function of the type of heterocycle involved:
flavonols, flavones, isoflavones, flavanones, anthocyanidins, and flavanols
(catechins and proanthocyanidins). In addition to this diversity, polyphenols
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
17
Introduction
may be associated with various carbohydrates and organic acids and with one
another [30].
The chemical structures of polyphenols and flavonoids are shown in the
figure below
The chemical structures of polyphenols
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
18
Introduction
The chemical structures of flavonoids
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
19
Introduction
As antioxidants, polyphenols may protect cell constituents against
oxidative damage and, therefore, limit the risk of various degenerative
diseases associated to oxidative stress. Experimental studies, in fact, strongly
support a role of polyphenols in the prevention of cardiovascular diseases,
cancer, osteoporosis, diabetes mellitus and neurodegenerative diseases [31].
In particular, it has been shown that the consumption of polyphenols limits
the development of atheromatous lesions, inhibiting the oxidation of low
density lipoprotein [32 - 35], which is considered a key mechanism in the
endothelial lesions occurring in atherosclerosis. However, many controlled
clinical trials have failed to demonstrate that increased antioxidants
consumption has a protective action against cardiovascular diseases [36].
For many years, polyphenols and other antioxidants were thought to protect
cell constituents against oxidative damage through scavenging of free
radicals. However, this concept now appears to be an oversimplified view of
their mode of action [37]. More likely, cells respond to polyphenols mainly
through direct interactions with receptors or enzymes involved in signal
transduction, which may result in modification of the redox status of the cell
and may trigger a series of redox-dependent reactions [38 - 40].
Both antioxidant and prooxidant [41] effects of polyphenols have been
described, with contrasting effects on cell physiologic processes: if as
antioxidants they improve cell survival, as pro-oxidant they may induce
apoptosis and block cell proliferation [42]. On the other hand, accumulating
evidence indicates that polyphenols might exert several other specific
biological effects such as the inhibition or reduction of different enzymes,
among which telomerase [43], cycloxygenase [44; 45], lipoxygenase [46; 47]
and the interaction with signal transduction pathways and cell receptors [48 50]. Moreover polyphenols can affect caspase-dependent pathways [51; 52],
cell cycle regulation [53] and platelet functions [54].
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
20
Introduction
For many years they received more and more attention as potential
therapeutic agents against several chronic degenerative diseases [55; 56], but
the complex relationships between antioxidant status and disease are still
poorly understood and have been studied intensively.
Resveratrol is a naturally occurring phytoalexin; its chemical name is
trans-3,5,4‟-trihydroxy stilbene. It occurs in two isoforms cis– and trans–
resveratrol, but trans–resveratrol is more biologically active than its cis–
isoform.
Resveratrol has been found in at least 72 plant species (distributed in 31
genera and 12 families), a number of which are included in the human diet,
such as mulberries, peanuts, and grapes. Grape skin is the main source of
resveratrol. Apart from these naturally occurring substances, red wine and
white wine also contain resveratrol. It is also synthesized in response to
environmental stressors that include water deprivation, UV irradiation and
especially fungal infections [57].
Resveratrol
during early nineties in the context of “French
paradox”; the phenomena wherein certain population of France, in spite of
eating a regular high fat diet, was less susceptible to heart diseases [58]. The
apparent cardioprotection was attributed to the regular consumption, in their
diet, of moderate doses of red wine rich in resveratrol [59].
In last few decades, resveratrol has gained the attention of scientists
worldwide; in fact,
have been the focus of
various in vivo and in vitro studies aimed at investigating its effect on
multiple pathophysiological processes and conditions. It has been reported to
possess anti-inflammatory [60], vasorelaxing [61] activity and it has been
demonstrated to inhibit lipid peroxidation [63; 64] and platelet aggregation,
which is a major contributor in the process of atherosclerosis [62], ex vivo.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
21
Introduction
Moreover resveratrol has been shown to possess potential anticancer activity
in various cancer cells at the initiation, promotion, and progression stages [65
- 67]. It is also well known to possess anti cancer properties in animal model
[68].
In the vast majority of cases, resveratrol displays inhibitory/stimulatory
effects
in
the
micromolar
range,
which
is
potentially
attainable
pharmacologically. It appears that resveratrol, as a pharmacological agent, has
a wide spectrum of targets[69].
Coumaric acids are organic compounds that are hydroxy derivatives of
cinnamic acid. There are three isomers, o-coumaric acid, m-coumaric acid,
and p-coumaric acid, that differ by the position of the hydroxy substitution of
the phenyl group. p-Coumaric acid is the most abundant isomer in nature.
Together with sinapyl alcohol and coniferyl alcohols, p-coumaric acid is a
major component of lignocellulose. It is biosynthesized from cinnamic acid
by the action of the P450-dependent enzyme 4-cinnamic acid hydroxylase.
p-Coumaric acid can be found in a wide variety of edible plants such as
peanuts, tomatoes, carrots, and garlic. It is a crystalline solid that is slightly
soluble in water, but well soluble in ethanol and diethyl ether. p-Coumaric
acid has antioxidant properties and is believed to reduce the risk of stomach
cancer [70] by reducing the formation of carcinogenic nitrosamines [71].
§ 1.5 ROS and cell signalling
Cells communicate with each other and respond to extracellular stimuli
through biological mechanisms called cell signalling or signal transduction
[18; 72]. Signal transduction is a process enabling information to be
transmitted from the outside of a cell to various functional elements inside the
cell. Signal transduction is triggered by extracellular signals such as
hormones, growth factors, cytokines and neurotransmitters [73].
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
22
Introduction
Signals sent to the transcription machinery responsible for expression of
certain genes are normally transmitted to the cell nucleus by a class of
proteins called transcription factors. By binding to specific DNA sequences,
these factors regulate the activity of RNA polymerase II. These signal
transduction processes can induce various biological activities, such as gene
expression, cell growth, muscle contraction and nerve transmission [74].
Although the endogenous generation of ROS are a consequence of
metabolic activities, many environmental stimuli including cytokines,
ultraviolet (UV) radiation, chemotherapeutic agents, hyperthermia and even
growth factors generate high levels of ROS that can perturb the normal redox
balance and shift cells into a state of oxidative stress. When the stress is
severe, survival is dependent on the ability of the cell to adapt to or resist the
stress, and to repair or replace the damaged molecules. Alternatively, cells
may respond to the insult by undergoing apoptosis, a process whereby
severely damaged cells are removed from the multicellular host, and which,
within limits, preserves the organism [75]. A number of stress response
mechanisms have evolved to help the cell and organism adapt to acute stress,
and acting in either a cooperative or antagonistic fashion they serve to
coordinate the acute cellular stress response and ultimately determine the
outcome. Many of these pathways have been faithfully preserved throughout
evolution. Among the main stress signalling pathways and/or central
mediators activated in response to oxidant injury are the extracellular signalregulated kinase (ERK), c-Jun amino-terminal kinase (JNK) and p38 mitogenactivated protein kinase (MAPK) signalling cascades, the phosphoinositide 3kinase (PI(3)K)/Akt pathway, the nuclear factor (NF)-kB signalling system,
p53 activation, and the heat shock response [75].
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
23
Introduction
Major signalling pathways activated in response to oxidative stress (from: Nature; Nov 9 2000;
408(6809):239-47. T. Finkel & N. J. Holbrook: Oxidants, oxidative stress and the biology of ageing).
Activation of these pathways is not unique to oxidative stress, as they are
known to have central roles in regulating cellular responses to other stresses
as well as regulating normal growth and metabolism. Indeed, in some
situations the response to oxidants may involve overstimulation of normal
ROS regulated signalling pathways. In general, the heat shock response, ERK,
PI(3)K/Akt and NF-kB signalling pathways exert a pro-survival influence
during oxidant injury, whereas activation of p53, JNK and p38 are more
commonly linked to apoptosis. However, numerous exceptions to these
generalities can be found.
Akt, also known as protein kinase B (PKB), is an evolutionarily
conserved serine/threonine kinase. Mammalian cells express three Akt
isoforms (Akt1–3) encoded by three separate genes. The amino acid
sequences of the three isoforms are almost identical; relative expression of
these isoforms, however, differs in various mammalian tissues. Akt is
activated by extracellular signals that activate PI3K. For instance, upon
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
24
Introduction
activation, growth factor receptors activate p110 (the catalytic subunit of
PI3K) either by recruiting p85 (the regulatory subunit of PI3K) or by
activating Ras, which can directly activate p110. Upon activation, p110
phosphorylates phosphoinositides (PI) at the D3 position of the inositol ring
to generate PI (3,4,5) P3 (PIP3). The rate-limiting step in Akt activation is the
binding of PIP3 to the pleckstrin homology (PH) domain of Akt and
subsequent translocation of Akt to the plasma membrane, where it is
phosphorylated on a threonine in the catalytic domain and a serine in the Cterminal regulatory domain. These modifications are required for full
activation. The kinase that phosphorylates the threonine is PDK1, while the
kinase that phosphorylates the serine was recently identified as mTORC2,
which is a complex containing the mammalian target of rapamycin (mTOR)
and Rictor [76]. Antagonizing PI3K activity can negatively regulate the
activity of Akt. PTEN (for „„phosphatase and tensin homolog deleted from
chromosome 10‟‟) is a 3 phosphoinositide phosphatase that negatively
regulates Akt activity by reducing the intracellular level of PIP3 produced by
PI3K [77]. Akt activity is also downregulated by activation of its downstream
effector mTORC1, which in turn induces a negative feedback mechanism that
inhibits Akt activity [78]. Hyperactivated Akt both provides protection from
apoptosis and promotes uncontrolled cell-cycle progression [79], two major
prerequisites for cancer susceptibility and this may explain, at least in part, its
frequent activation in human cancers [78]. However, the principal role of Akt
is to facilitate growth factor-mediated cell survival and to block apoptotic cell
death, which is achieved by phosphorylating and deactivating proapoptotic
factors such as BAD, caspase-9, and murine double minute-2 (MDM2) [80].
Akt also phosphorylates and inactivates glycogen synthase kinase-3 (GSK3), the inactivation of which prompts upregulation of cyclin D and enhances
cell cycling. Akt is regulated by oxidative stress for cell survival, and
phosphorylates IkB kinase-a/b. Activated IkB kinase-a/b, in turn, causes
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
25
Introduction
activation and nuclear translocation of NF-kB-dependent prosurvival genes
[80].
Akt Pathway (from: www.invitrogen.com)
The MAPKs, instead, comprise a family of ubiquitous prolinedirected,
protein-serine/threonine kinases, which play an essential role in sequential
transduction of biological signals from the cell membrane to the nucleus [81].
In mammalian cells, there are three well-defined subgroups of MAPKs: the
extracellular signal regulated kinases (ERKs, including ERK-1 and ERK-2
isoforms), the c-Jun N-terminal kinases (JNKs, including JNK-1, JNK-2, and
JNK-3 isoforms), and the p38 MAPKs (including p38-α, p38-β, p38-γ, and
p38-δ isoforms). Each subgroup of MAPKs is activated through a cascade of
sequential phosphorylation events, beginning with the activation of MAPK
kinase kinases (MAP3Ks). The MAP3Ks phosphorylate and activate a
downstream dualspecificity MAPK kinases (MAP2Ks), which in turn
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
26
Introduction
stimulate MAPK activity through dual phosphorylation on threonine and
tyrosine residues within a conserved tripeptide motif [81; 82]. The three
subgroups of MAPKs (i.e., ERKs, JNKs, and p38 MAPKs) are involved in
both cell growth and cell death, and the tight regulation of these pathways is
paramount in determining cell fate [83]. The deleterious consequences of
sustained activation of MAPK pathways may include excessive production of
MAPK-regulated genes, uncontrolled proliferation, and unscheduled cell
death.
Studies have demonstrated that ROS can induce or mediate the
activation of the MAPK pathways [84]. A number of cellular stimuli that
induce ROS production also in parallel can activate MAPK pathways in
multiple cell types. The prevention of ROS accumulation by antioxidants
blocks MAPK activation after cell stimulation with cellular stimuli [84; 85],
indicating the involvement of ROS in activation of MAPK pathways.
Moreover, direct exposure of cells to exogenous H2O2, to mimic oxidative
stress, leads to activation of MAPK pathways [86].
MAPK cascades (from: J Signal Transduct.; 2011:792639. Y. Son, et al.: Mitogen-Activated Protein
Kinases and Reactive Oxygen Species: How Can ROS ActivateMAPK Pathways?).
The initiating events leading to activation of pathways in response to
oxidants are incompletely understood. Although a large number of signaling
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
27
Introduction
pathways appear to be regulated by ROS, the signaling molecules targeted by
ROS are less clear. There is growing evidence, however, that redox regulation
might occur at multiple levels in the signaling pathways from receptor to
nucleus. Receptor kinases and phosphatases themselves may be targets of
oxidative stress. Growth factor receptors are most commonly activated by
ligand-induced dimerization or oligomerization that autophosphorylates its
cytoplasmic kinase domain. Ligand-independent clustering and activation of
receptors in response to ultraviolet light have also been well demonstrated,
and this effect appears to be mediated by ROS [18].
Oxidants seem to activate the ERK and the PI(3)K/Akt pathways largely
through stimulation of growth-factor receptors, mimicking the actions of
natural ligands. Many growth-factor receptors have been shown to undergo
enhanced phosphorylation in response to direct treatment with oxidants, and
agents or conditions that prevent receptor phosphorylation likewise inhibit the
activation of ERK and Akt by oxidants [75]. One mechanism proposed to
explain this effect is oxidant-mediated inactivation of critical phosphatases
necessary for dephosphorylation (turning off) of the growth-factor receptors.
Support for such a mechanism has come from the finding that hydrogen
peroxide, either derived exogenously or produced endogenously after growthfactor stimulation, can reversibly inactivate protein-tyrosine phosphatase 1B
in cells. The activation of growth-factor-receptor signalling pathways by
oxidants is consistent with the demonstration that low concentrations of
exogenous hydrogen peroxide are mitogenic [75].
Oxidative stress might induce activation of the JNK and p38 kinase pathways
by an additional mechanism. The redox regulatory protein thioredoxin (Trx)
has been shown to bind to apoptosis signal-regulating kinase (ASK1), an
upstream activator of both JNK and p38, and under normal conditions inhibit
its activity. However, oxidative stress causes dissociation of the Trx–ASK1
complex and subsequent activation of the downstream JNK and p38 kinase.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
28
Introduction
Similarly, biochemical evidence indicates that under non-stressed conditions
glutathione S-transferase binds to JNK to inhibit its activation, but that this
interaction is also disrupted by oxidative stress [75].
These results show an intimate coupling between alterations in the
intracellular redox state and the activity of downstream stress-activated
pathways. The observation that multiple pathways are sensitive to a rise in
ROS levels indicates that these pathways may have evolved, in part, to allow
organisms to survive within an aerobic environment. In addition, it suggests
that a rise in ROS might represent a common, if not universal, signal of
cellular stress.
§ 1.6 ROS and apoptosis
Although under normal conditions there is a balance between ROS
formation and antioxidants, in several pathological scenarios the antioxidant
defences become insufficient resulting in oxidative stress leading often to
apoptosis and cell death. Apoptosis (or programmed cell death) is the
mechanism used by mammals, plants and other organisms to eliminate
redundant or damaged cells [87]. Cells die in response to a variety of stimuli
and during apoptosis they do so in a controlled, regulated fashion. This
makes apoptosis distinct from another form of cell death called necrosis in
which uncontrolled cell death leads to lysis of cells, inflammatory responses
and, potentially, to serious health problems. Apoptosis, by contrast, is a
process in which cells play an active role in their own death (which is why
apoptosis is often referred to as cell suicide). Upon receiving specific signals
instructing the cells to undergo apoptosis a number of distinctive changes
occur in the cell. A family of proteins known as caspases are typically
activated in the early stages of apoptosis. These proteins breakdown or
cleave key cellular components that are required for normal cellular function
including structural proteins in the cytoskeleton and nuclear proteins such as
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
29
Introduction
DNA repair enzymes. The caspases can also activate other degradative
enzymes such as DNases, which begin to cleave the DNA in the nucleus
[88]. Apoptotic cells display distinctive morphology during the apoptotic
process. Typically, the cell begins to shrink following the cleavage of lamins
and actin filaments in the cytoskeleton. The breakdown of chromatin in the
nucleus often leads to nuclear condensation and in many cases the nuclei of
apoptotic cells take on a "horse-shoe" like appearance. Cells continue to
shrink, packaging themselves into a form that allows for their removal by
macrophages. These phagocytic cells are responsible for clearing the
apoptotic cells from tissues in a clean and tidy fashion that avoids many of
the problems associated with necrotic cell death. In order to promote their
phagocytosis by macrophages, apoptotic cells often ungergo plasma
membrane changes that trigger the macrophage response. One such change
is the translocation of phosphatidylserine from the inside of the cell to the
outer surface. The end stages of apoptosis are often characterised by the
appearance of membrane blebs or blisters process. Small vesicles called
apoptotic bodies are also sometimes observed [88]. In some cases the
apoptotic stimuli comprise extrinsic signals such as the binding of death
inducing ligands to cell surface receptors called death receptors. These
ligands can either be soluble factors or can be expressed on the surface of
cells such as cytotoxic T lymphocytes. The latter occurs when T-cells
recognise damaged or virus infected cells and initiate apoptosis in order to
prevent damaged cells from becoming neoplastic (cancerous) or virusinfected cells from spreading the infection. Apoptosis can also be induced by
cytotoxic T-lymphocytes using the enzyme granzyme.
In other cases apoptosis can be initiated following intrinsic signals
that are produced following cellular stress. Cellular stress may occur from
exposure to radiation or chemicals or to viral infection. It might also be a
consequence of growth factor deprivation or oxidative stress caused by free
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
30
Introduction
radicals. In general intrinsic signals initiate apoptosis via the involvement of
the mitochondria. Besides serving as the major energy producer,
mitochondria also play a critical role in the regulation of apoptosis, and act
as a major switch to initiate apoptosis in mammalian cells [89]. They contain
many pro-apoptotic proteins such as Apoptosis Inducing Factor (AIF),
Smac/DIABLO and cytochrome c. Although apoptosis can occur in the
absence of cytochrome c release, cytochrome c directly activates
downstream effectors when injected into the cytosol in the absence of
upstream signals. Release may occur secondary to the onset of
mitochondrial permeability transition (MPT) pore, which is permeable to
solutes of less than 1200 Da [90]. The onset of MPT pore leads to loss of
mitochondrial membrane potential and to swelling of the matrix space with
eventual disruption of mitochondrial membranes and release of cytochrome
c [91]. This catastrophic event is caused by oxidative damage to
mitochondria in concert with mitochondrial calcium overload. Therefore, an
increased mitochondrial formation of ROS triggers the intrinsic pathway by
increasing the permeability of the outer mitochondrial membrane through
the opening of transition pores. The opening of the permeability transition
pore is favoured by oxidative stress through oxidation of intracellular
glutathione and other critical sulfhydryl groups [87]. As a result of this
process, cytochrome c moves from the intermembrane space into the cell‟s
cytoplasm where it joins another factor (Apaf-1). In the presence of dATP
this complex polymerizes into an oligomer known as „apoptosome‟. The
apoptosome activates a protease (caspase-9), which in turn activates
caspase-3. The cascade of proteolytic reactions also activates DNAses and in
the end the process results in cell death. Under normal conditions, various
anti-apoptotic factors (including Bcl-xL) prevent the mitochondrial
permeability transition as long as they remain bound to the outer membrane
[87]. This factor is eliminated when another factor, Bax, is translocated to
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
31
Introduction
mitochondria, starting apoptosis. The gradual loss of cytochrome c from the
intermembrane space during apoptosis favours the mitochondrial formation
of O2−• in two ways: (1) cytochrome c is a scavenger of O2−• and (2) as
cytochrome c is released, the respiratory chain becomes more reduced
because electron flow between Complex III and Complex IV slows down
[87].
ROS appear to be mitochondria derived and responsible for later
mitochondrial events leading to full activation of the caspase cascade. How
ROS acts in this scenario is not entirely understood. Oxidation of the
mitochondrial pores by ROS may contribute to cytochrome c release due to
disruption of the mitochondrial membrane potential. In contrast, it is unclear
how the initial ROS is released from mitochondria. If a sequential event is
postulated, initial released ROS could directly or indirectly (via ceramide
generation) increase the gating potential of the pore. Taken together, it
seems that mitochondria are both source and target of ROS.
Illustration of the main apoptotic signalling pathways involving mitochondria (from:
www.sgul.ac.uk/dept/immunology/~dash. Apoptosis. Phil Dash. Basic Medical Sciences,
St.George‟s, University of London.).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
32
Aim of the work
CHAPTER 2
AIM OF THE WORK
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
33
Aim of the work
The deleterious effects of free radicals have been known for almost 50
years. More recently, however, an essential role for free radicals in
physiologic control of several aspects of cell function has been demonstrated.
Free radicals are, indeed, now considered as key regulatory molecules vital
for life, but they cause cellular and organ damage when produced in excess or
when innate antioxidant defenses are overwhelmed [30].
A variety of pathogenic stimuli can increase ROS production within the
endothelial cell (EC) triggering biochemical and cellular processes, such as
apoptosis and proliferation that eventually result in endothelial dysfunction.
This phenomenon represents the initial step in the progression toward
pathological syndromes, such as atherosclerosis and hypertension [92].
In this context, it has been hypothesized that Natural Antioxidants (NA)
consumption, can counteract the effects of ROS by preventing ROS-induced
oxidative damage and preserve endothelial function reducing the occurrence
of cardiovascular events [93]. In fact their consumption has been associated
with a reduced incidence of risk factors for cardiovascular diseases (CVD)
[36].
CVD is of multifactorial etiology associated generally to a variety of
risk
factors
for
its
development
including
hypercholesterolaemia,
hypertension, smoking, diabetes, poor diet, stress and physical inactivity
among others. During the last few decades, research data has prompted a
passionate debate as to whether oxidation, or specifically, oxidative stress
mediated by free radicals/ROS/RNS, is a primary or secondary cause of many
chronic diseases. As a result, scientific resources have focused to a large
extent on the role that antioxidants could play to delay or prevent oxidative
stress and consequently the incidence of chronic disorders.
The endothelium is a complex organ system that controls the
homeostasis of the vasculature by integrating signals between the vascular
wall and the vessel lumen. Under physiological conditions, it maintains a
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
34
Aim of the work
normal vascular tone and blood fluidity by elaborating a variety of factors,
such as nitric oxide, prostacyclin and endothelin. However, in pathological
situations the endothelium can also modify its phenotype facilitating
vasoconstriction, inflammation and thrombotic events. These abnormal
responses manifest in different clinical settings, such as hypercholesterolemia,
hypertension, diabetes mellitus, and occur in the absence of any
morphological change of the vessel.
The etiology of these altered endothelial functions is multi-factorial and the
mechanisms underlying them are complex and not yet fully elucidated.
Today, there is substantial evidence that many endothelial functions are
sensitive to the presence of reactive oxygen species and subsequent oxidative
stress.
The widely accepted notion that consumption of antioxidants is useful
to counteract oxidative stress and promote good health is at the basis of
popular antioxidant rich diets and supplementations.
However, the clinical use of exogenous antioxidant substances has generally
failed to live up to their early promise; in fact many controlled clinical trials
have failed to demonstrate that increased NA consumption has a protective
action against CVD [36].
The reason for these disappointing findings is unclear, but some possible
explanations have been proposed. In the endothelium, ROS are not only
involved in pathological processes but, by modulating redox-regulated
intracellular signals, they also finely tune EC physiology [94].
It has been proposed that ROS might function as dual effectors modulating
both prosurvival and antisurvival signals [95]. Strategies aimed at the
suppression of all ROS signaling could have the unexpected consequences of
negatively impacting endothelial function. In addition, NA can have a prooxidant effect under particular conditions, paradoxically increasing ROS
production and resulting in cell damage [96].
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
35
Aim of the work
In this light, antioxidant-based strategies based on the suppression of all
intracellular ROS, to prevent endothelial damage, may have the untoward
effect of leading to EC loss.
While there is a consistent body of literature on the protective effects of NA
against diseases or toxic drugs, there are relatively few reports on their
possible toxicity. Indeed, NA-associated cardiovascular effects appear to be
concentration dependent [97] and the molecular mechanisms underlying this
phenomenon, as well as the associated outcomes in vascular cells, are largely
unknown.
Therefore, the purpose of the research was to assess how natural
antioxidant, such as resveratrol and coumaric acid, affect differently the
(patho)physiology of endothelial cells extracted from umbilical cord
(HUVEC).
We initially evaluated the change of intracellular levels of ROS in presence of
NA. Furthermore, using inhibitors of specific enzyme responsible of ROS
production, the potential source of these molecules has been investigated.
To further elucidate the molecular basis of NA-induced oxidative stress, we
sought to identify signaling transduction pathways responsible for the
observed changes in the cellular response; so we studied specific pathways
that regulate proliferation, apoptosis and cell cycle. In particular, we tried to
assess whether AKT pathway could regulates cell survival in response to NAinduced oxidative stress.
Finally we investigated the molecular mechanism underlying the observed
phenomena, considering the involvement of a possible mitochondrial damage
in response to intracellular ROS produced after cells treatment with NA.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
36
Material and Methods
CHAPTER 3
MATERIALS AND METHODS
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
37
Material and Methods
§ 3.1. Reagents.
Coumaric acid, resveratrol, rotenone, Angiotensin II, insulin, and N-acetyl
cysteine
(NAC)
were
supplied
by
Sigma
(St
Louis,
MO).
Diphenyleneiodonium (DPI), Wortmannin, LY-294002, and Staurosporine
were from Calbiochem (San Diego, CA).
§ 3.2 Cell culture and treatments.
Human umbilical vein ECs (Cell Applications, San Diego, CA) were cultured
in EC basal medium (Cell Applications) supplemented with Endothelial Cell
Growth Supplement (Cell Applications). When confluent, ECs were
subcultured at a split ratio of 1:2 and used within three passages. In order to
mimic physiological vessel wall conditions, before experimentation, cells
were grown until confluence to reach contact-dependent growth inhibition.
Unless not specified in the text, cells were plated in 96-well black plates (BD
Falcon, Franklin Lakes, NJ) and processed for experiments in EC-defined
medium (European Collection of Cell Cultures, Salisbury, UK).
Intracellular ROS measurements, immunoblotting, and protein carbonylation
assays were performed after 80 min, while cell viability, metabolic assays,
and apoptosis were done after 4 h. Dose- and time-dependent immunoblot
experiments were performed as indicated in figure legends, after culturing
ECs for 12 h under serum-free condition.
In selected experiment, cells were preincubated for 30 min with the Akt
activator insulin and the specificity of insulin-mediated Akt activation was
demonstrated by using the selective phosphoinositide-3 kinase (PI3K)
inhibitors Wortmannin (20nM) and LY-294002 (10µM) [98].
To study the contribution to protein carbonylation and ROS levels of flavincontaining oxidases, we employed the flavoproteins inhibitor DPI (10µM)
[99]. Staurosporine treatment (1µM) was used to induce EC apoptosis [100].
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
38
Material and Methods
As an index of NADH- and NADPH-dependent oxidase activity, we assessed
treatment-induced variations of NADH and NADPH consumption [101]. DPIsensitive flavin oxidase activity was assessed as previously reported [102].
§ 3.3 Measurements of intracellular ROS.
Intracellular ROS levels were determined by using the ROS molecular probe
2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA) (Molecular Probe,
Eugene, OR) as previously described with minor modification [103]. Within
the cell, esterases cleave the acetate groups on H 2DCF-DA, thus trapping the
reduced form of the probe (H2DCF). Intracellular ROS oxidize H2DCF,
yielding the fluorescent product, DCF.
After treatments, cells were incubated for 30 min with Hanks‟ Balanced Salt
Solution (HBSS) containing 5µM H2DCF-DA, then washed twice with HBSS
and fluorescence was measured by using a GENios plus microplate reader
(Tecan, Männedorf, CH). Excitation and emission wavelengths used for
fluorescence quantification were 485 and 535 nm, respectively. All
fluorescence measurements were corrected for background fluorescence and
protein concentration. Using untreated cells as a reference, the antioxidant and
prooxidant outcome was evaluated by comparison of five measurements and
expressed as a percentage of untreated controls.
§ 3.4 Measurements of protein carbonylation.
Proteins are one of the major targets of oxygen free radicals and other reactive
species. Oxidative modification of proteins modifies the side chains of
methionine, histidine, and tyrosine and forms cysteine disulfide bonds. Metal
catalyzed oxidation of proteins introduces carbonyl groups (aldehydes and
ketones) at lysine, arginine, proline or threonine residues in a site-specific
manner.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
39
Material and Methods
Protein carbonyl groups were measured with the OxyBlot protein oxidation
detection kit (Chemicon, Temecula, CA), following the protocol provided by
the manufacturer.
In brief, proteins (20 µg) were denatured with 12% sodium dodecylsulfate
(SDS), derivatized to 2,4-dinitrophenylhydrazone (DNPhydrazone) by
reaction
with
2,4-dinitrophenylhydrazine (DNPH)
then
mixed
with
neutralization solution and mercaptoethanol. To evaluate the selectivity of
carbonyl measurements, some protein samples underwent the protein
carbonyl detection procedure without the derivatization step (negative
control).
DNP-derivatized proteins were electrophoresed through a reducing 12% SDSpolyacrylamide gel and electroblotted onto a nitrocellulose membrane.
The membrane was blocked with 1% bovine serum albumin (BSA) for 1 h at
room temperature and incubated overnight at 4°C with rabbit anti-DNP
antibody (1:500). The levels of carbonylated proteins were detected with
horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2000) for 1
h at room temperature.
Blots were developed by an enhanced chemiluminescence System
(Amersham, Buckinghamshire, UK) and densitometric analyzed by using the
Versadoc Imaging System (Bio-Rad, Hercules, CA). Individual densitometric
results were normalized to -actin immunoreactivity and results were
expressed as a percentage of untreated controls.
§ 3.5 Measurement of NADH and NADPH consumption.
The rate of oxidation of NADH and NADPH in the presence of oxygen
(oxidase activity) was measured spectrophotometrically using methods
similar to those previously described [101].
After treatments, confluent human umbilical vein endothelial cells (HUVEC)
were scraped off from the flasks in 300 µl of 50mM phosphate buffer, pH 7.4,
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
40
Material and Methods
plus protease and phosphatase inhibitors. The cell suspension was sonicated
for three cycles of 20 s on ice; the homogenate was assessed for protein
content and stored on ice until use. The assay was performed in 200 µl of
40mM Tris-Mes buffer (pH 7.4) containing either 250 µM NADH or 250 µM
NADPH.
The reaction was started by adding 50 µl of each sample in the reaction mix
and the rate of NADH or NADPH consumption was monitored during 30 min
by reading the decrease in absorbance at 340 nm using a GENios plus
microplate reader (Tecan). The extinction coefficient used to calculate the
amount of NADH/NADPH consumed was 3.732 * 10−3 ml/nmol, which
results from the microplate reader path length for a reaction volume of 200 µl
in a 96-well plate.
For measurements of specific flavin oxidase activity, the rate of NADH or
NADPH consumption inhibitable by DPI, a flavoproteins inhibitor, was used
[99]. This was done by adding DPI (10µM) 15 min prior to treatments.
The „„DPI-inhibitable‟‟ NADH/NADPH consumption was used as a measure
of flavin-containing NADH or NADPH oxidase activity [102]. All
measurements were corrected for protein content, and results were expressed
as nanomoles per minute per milligram of protein.
§ 3.6 Cell viability assay.
Cell viability was assessed by using propidium iodide (PI), Hoechst 33342
double fluorescent staining. PI can only enter cells with disrupted membrane
integrity and therefore stains nonviable cells. Thus, all cell nuclei could be
recognized by the blue fluorescence of Hoechst, while nuclei of damaged
cells fluoresced red due to the accumulated PI. After treatments, cells were
stained with PI and Hoechst (10 µg/ml) (Invitrogen, Carlsbad, CA), washed
with PBS, and fluorescence was measured by using a GENios plus microplate
reader (Tecan). Excitation and emission wavelengths used for fluorescence
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
41
Material and Methods
quantification were, respectively, 340 and 485 nm for Hoechst and 485 and
612 nm for PI. Results were expressed as a percent of untreated control cells.
§ 3.7 Cell metabolic assay.
Cell metabolic activity was assessed in 96-well plates (BD Falcon) by using
the
colorimetric
3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium
bromide (MTT) assay (Promega, Madison, WI).
Yellow MTT enters the cells and passes into the mitochondria where it is
reduced to an insoluble, coloured (dark purple) formazan product.
This reduction takes place only when mitochondrial reductase enzymes are
active, and therefore conversion can be directly related to the number of
viable (living) cells.
Mitochondrial dehydrogenases of viable cells cleave the tetrazolium ring,
yielding purple MTT formazan crystals which are insoluble in aqueous
solutions. The crystals can be dissolved in acidified isopropanol. The
resulting purple solution is spectrophotometrically measured. An increase in
cell number results in an increase in the amount of MTT formazan formed
and an increase in absorbance.
So after treatments, cells were added with 20 µl MTT solution (5 mg/ml) in
medium M199 and incubated at 37°C in a cell incubator for 60 min. At the
end of the incubation period, the medium was removed and the cell
monolayer was washed twice with HBSS. The converted dye was solubilized
with acidic isopropanol (0.04N HCl in absolute isopropanol), and plates were
analyzed at 570 nm using a GENios plus microplate reader (Tecan) with
background subtraction at 650 nm.
Results were expressed as a percent of untreated control cells.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
42
Material and Methods
§ 3.8 Cell apoptosis assay.
Cell apoptosis was assessed after treatments by using the fluorimetric kit
APOPercentage (Biocolor Ltd, Carrickfergus, UK), following the protocol
provided by the manufacturer. The assay has been used with several adherent
cell lines including HUVEC [104].
The assay uses the dye 3,4,5,6,-tetrachloro-2‟,4‟,5‟,7‟-tetraiodofluorescein
that is selectively imported by cells that are under going apoptosis.
Maintaining the asymmetric composition is an energy dependant process
involving the activity of enzymes, termed „flippases‟. In apoptotic committed
cells flippase regulation is either overwhelmed, or is inactivated by the
activity
of
the
enzyme
„scramblase‟
(floppase).
Exposure
of
phosphatidylserine to the exterior surface of the membrane has been linked to
the onset of the execution phase of apoptosis. The transfer of
phosphatidylserine to the outside of the membrane permits the transport of the
APOPercentage dye into the cell. The uptake of the dye is uni-directional,
leading to dye accumulation within the cell. As the cell shrinks in volume,
during the apoptotic process, the cell dye content becomes more concentrated.
Confluent cells, plated in 96-well black plates (BD Falcon), were treated as
described in § 3.2 of the „„Materials and Methods‟‟ section.
At the end of treatments, the APOPercentage dye was added to each well
(dilution 1:10) and cells incubated for 30 more min at at 37°C in a 5% CO2
incubator. After thoroughly washing, 100 µl of APOPercentage dye release
reagent was added to each well, and the cell-bound dye recovered into
solution was measured using a GENios plus microplate reader (Tecan) with
excitation and emission of 530 and 580 nm, respectively.
Results were expressed as a percent of untreated control cells.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
43
Material and Methods
§ 3.9 Immunoblotting analysis.
Cells were cultured in T25 culture flasks (BD Falcon), treated as indicated in
§ 3.2 of the „„Materials and Methods‟‟ section and then processed for
immunoblotting as previously reported [105].
Sample
proteins
were
separated
using
SDS
polyacrylamide
gel
electrophoresis (SDS-PAGE) at 200 V for 45 minutes. Then they were
transfered onto nitrocellulose membrane using electroblotting (100V for 1h).
After to protein immobilization the membrane was blocked with TBS
containing 5% non-fat powdered milk and 0.1% Tween-20 (blocking buffer)
for 1 hour, on the shaker, at room temperature.
So the nitrocellulose membrane was incubated with primary antibody in
blocking buffer overnight at 4°C.
We used specific antibodies against the total and phosphorylated form of the
protein kinase Akt, the mitogen-activated protein kinases (MAPKs) p42/
44MAPK and stress-activated protein kinase/Jun N-terminal kinase (SAPK/
JNK)MAPK, and the cleaved and uncleaved form of caspase-3 (Cell
Signaling, Danvers, MA).
The next day, after thoroughly washing in TBS containing 0.1% Tween-20,
the nitrocellulose membrane was incubated with the secondary antibody
solution in blocking buffer for 1 hour, on the shaker, at room temperature.
Densitometric analysis was performed by using the Versadoc Imaging System
(Bio-Rad) to scan the signals. Results were expressed as arbitrary units, and
ratios of individual densitometric results were normalized to -actin
immunoreactivity.
§ 3.10 MMP assay
Mitochondrial membrane potential was assessed after treatments by using the
fluorimetric kit JC-1 Mitochondrial Membrane Potential Detection Kit
(Biotium, Inc. USA), following the protocol provided by the manufacturer.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
44
Material and Methods
JC-1 Assay Kit uses a cationic dye (5,5‟,6,6‟-tetrachloro-1,1‟,3,3‟tetraethylbenzimidazolylcarbocyanine
iodide)
to
signal
the
loss
of
mitochondrial membrane potential [106].
The loss of mitochondrial membrane potential (ΔΨ) is a hallmark for
apoptosis. It is an early event preceding phosphatidylserine externalization
and coinciding with caspase activation.
In healthy cells, the dye stains the mitochondria bright red. The negative
charge established by the intact mitochondrial membrane potential allows the
lipophilic dye, bearing a delocalized positive charge, to enter the
mitochondrial matrix where it accumulates. When the critical concentration is
exceeded, J-aggregates form, which become fluorescent red. In apoptotic
cells, the mitochondrial membrane potential collapses, and the JC-1 cannot
accumulate within the mitochondria. In these cells JC-1 remains in the
cytoplasm in a green fluorescent monomeric form. Apoptotic cells, showing
primarily green fluorescence, are easily differentiated from healthy cells
which show red and green fluorescence.
Confluent cells, plated in 96-well black plates (BD Falcon), were treated as
described in § 3.2 of the „„Materials and Methods‟‟ section.
At the end of treatments, the JC-1 dye was added to each well and cell
incubated for 15 min at 37°C in a 5% CO2 incubator.
After thoroughly washing, 100 µl of PBS was added to each well, and red
fluorescence (excitation 550 nm, emission 600 nm) and green fluorescence
(excitation 485 nm, emission 535 nm) were measured using a GENios plus
microplate reader (Tecan) with excitation and emission of 530 and 580 nm,
respectively.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
45
Material and Methods
§ 3.11 Statistical analysis.
Data are expressed as means ± SDs of four or five different experiments. Oneway ANOVA followed by a post hoc Newman- Keuls Multiple Comparison
Test were used to detect differences of means among treatments with
significance defined as p < 0.05. When appropriate, two-way ANOVA with a
Bonferoni post test was used to assess any differences among the treatments
and the times (p < 0.05). Statistical analysis was performed using GraphPad
Prism version 5.00 for Windows (GraphPad Software, San Diego, CA).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
46
Results
CHAPTER 4
RESULTS
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
47
Results
§ 4.1 Dose-dependent effect of NA on EC ROS levels and protein
carbonylation
The study began with the quantification of ROS levels in endothelial
cells (ECs) treated with various concentrations of two NA (Resveratrol and
Coumaric Acid) and untreated cells (CTRL) used as control.
Intracellular ROS generation was examined in ECs in response to NA using
2‟,7‟-dichlorodihydrofluorescein diacetate (H2DCF-DA). This probe enters
the cells and can be oxidized in the presence of ROS, generating the
fluorescent compound, DCF.
First of all we validated this ROS assay treating ECs with increasing
doses of H2O2 and Angiotensin II to test their capability to detect variations of
intracellular ROS levels in response to these well-known pro-oxidants in the
used cellular model [107; 108].
Data reported in figure 1A show that the probe has a significant dynamic
range and responds linearly to increasing doses of H 2O2 and Angiotensin II,
confirming the test validity.
Then to determine the effects of NA on ECs, cells were treated as
indicated previously and intracellular ROS levels were assessed after
treatments. This was evaluated as change in DCF fluorescence. The results
from five pooled measurements are shown below and results are expressed as
percentage of untreated controls.
Treatment of ECs with 0.5µM resveratrol exerted a significant antioxidant
effect confirming the protective role of NA previously described and further
validating our experimental system [109; 110]. However, the exposure of cell
cultures to higher concentrations of resveratrol increased intracellular ROS
levels in a dose-dependent manner. As a result, the antioxidant effect seen at
0.5µM was lost and a marked pro-oxidant effect was evident at both 10 and
25µM (fig. 1B).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
48
Results
Coumaric acid treatment resulted in a similar pattern with a marked
prooxidant effect starting at 10µM and persisting at 25µM (fig. 1C).
We next investigated whether the combination of low doses of the two
tested compounds had a synergistic or additive effect on intracellular ROS
levels. Using the two NA together (fig. 1D) no differences, on intracellular
ROS levels, were detected versus the same concentration (0.5 and 5µM) of an
antioxidant alone.
To gain further information on the antioxidant and pro-oxidant effect of
these two compounds, we assessed variations of the protein carbonyl content
in response to NA treatment. In fact carbonylation of proteins is considered a
widespread indicator of oxidative damage and disease-derived protein
dysfunction [111].
Protein carbonyl groups were measured with the OxyBlot protein oxidation
detection kit (Chemicon, Temecula, CA), following the protocol provided by
the manufacturer as described in Material and Methods.
Figures 1E and 1F show as the protein carbonylation pattern elicited by both
NA strictly overlapped that of ROS, strongly confirming the antioxidant and
pro-oxidant effect exerted by the tested compounds. Representative western
blottings of protein carbonylation experiments are reported in figure 2.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
49
Results
Fig. 1. Confluent ECs were stimulated for 80 min as indicated in figure.
Intracellular ROS levels were assessed after treatments as described in the „„Materials and
Methods‟‟ section. (A) Dose-response effect of H2O2 and Angiotensin II on intracellular ROS
levels. (B and C) Intracellular ROS levels in cultured ECs in the absence (CTRL) or presence of the
indicated concentration of (B) resveratrol and (C) coumaric acid. (D) Effect of single or combined
dose of NA on intracellular ROS generation.
(E and F) Measurement of protein carbonylation in cultured ECs in the absence (CTRL) or
presence of the indicated concentration of (E) resveratrol and (F) coumaric acid. Protein
carbonylation was assessed after treatments as reported in the „„Materials and Methods‟‟ section.
Graphs represent the immunodensity quantitative analysis of three different immunoblot
experiments. (Individual densities from the different bands were added up to generate one single
value.)
CTRL, untreated cells; R, resveratrol; C, coumaric acid; H2O2, hydrogen peroxide; and Ang II,
Angiotensin II. Data are expressed as percent of control. (A–F)
*Significantly different from the control, §significantly different from each other, and #
significantly different from each other (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
50
Results
Fig. 2. Representative western blottings of protein carbonylation experiments
§ 4.2 NA dose dependently induce ECs impairment
To investigate how the ROS induced by NA, affect the physiology of
human endothelial cells, we measured cell viability, metabolic activity and
apoptosis.
Cell viability was assessed by using propidium iodide (PI), Hoechst
33342 double fluorescent staining. as described in Material and Methods.
Exposure of cell cultures to increasing concentrations of resveratrol and
coumaric acid decreased cell survival (figs. 3A and 3B), an effect consistent
with the increase in ROS levels and protein carbonyl content.
It was observed that treatment of EC with 0.5 and 5 μM of NA did not induce
obvious changes in cell viability compared to untreated cells. In contrast,
treatment of cells with higher concentrations (10, 25 μM) of resveratrol and
coumaric acid induced a statistically significant reduction in cell viability
compared to the controls.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
51
Results
Then we evaluated apoptosis using the fluorimetric kit APOPercentage
(Biocolor Ltd, Carrickfergus, UK), following the protocol provided by the
manufacturer as described in Material and Methods. The cells were treated
with different concentrations of NA and we used, as positive control, cells
treated with staurosporine (1µM), a known inducer of apoptosis. As can be
seen in fig. 3B, the NA induced an increase in apoptosis at higher
concentrations, but they had no effect at lower concentrations; these results
confirm those already obtained in the previous experiments on the
measurement of ROS and cell viability.
However, while viability decreased dose dependently (fig. 3A),
apoptosis did not increase in a dose-dependent fashion (fig. 3B), suggesting a
potential shift toward a necrotic mechanism at high NA concentrations.
However, the involvement of apoptosis was confirmed by increased levels of
cleaved (active form) caspase-3 immunoreactivity (fig. 4).
Consistent with the observed cell damage, a significant decrease in cell
metabolic activity, was induced by both 10 and 25µM of NA (fig. 3C).
Importantly, the treatment of ECs with 0.5 and 5µM of NA failed to induce
any evident variation in the Hoechst/PI fluorescence ratio or apoptotic rate as
compared to untreated cells (figs. 3A and 3B) excluding the possibility of a
general toxic effect of NA in our experimental system. Rather, consistent with
the reported antioxidant effect, an increase of metabolic activity and a
significant antiapoptotic effect were detected at the dose of 0.5µM (figs. 3B
and 3C).
We next ascertained the causative role of ROS in NA-induced protein
carbonylation and ECs damage by using the ROS scavenger N-acetyl-cysteine
(NAC). Pretreatment of cell cultures with 5mM NAC prevented both NAinduced protein carbonylation and ECs damage (figs. 5A–C).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
52
Results
Fig. 3. Confluent ECs were stimulated for 4 h as indicated in figure. Cell viability, metabolic
activity, and apoptosis were assessed after treatments as reported in the „„Materials and Methods‟‟
section. Staurosporine at concentration of 1µM was used to induce ECs apoptosis. (A)
Quantification of Hoechst/PI ratio, (B) apoptosis, and (C) metabolic activity in cultured ECs in the
absence (CTRL) or presence of the indicated NA concentration. CTRL, untreated cells; R,
resveratrol; C, coumaric acid; and Stauro, staurosporine. Data are expressed as percent of
maximum. (A–C) *Significantly different from the control, §significantly different from each
other, #significantly different from each other, and •significantly different from all values (p <
0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
53
Results
Fig.4. Confluent ECs were stimulated for 80 minutes as indicated in figure and then processed for
immunoblotting as described in material and methods. The upper part of the figure shows the
immunoreactivity of cleaved (active form) and uncleaved (non-active form) caspase-3, while the
lower part reports the immunodensity quantitative analysis. Immunodensity values (cleaved and
uncleaved) are represented on different graphs sharing the same y-axis. CTRL; untreated cells. R;
resveratrol. C; coumaric acid. Stauro; staurosporine. Ratios of individual densitometric results were
normalized to -actin immunoreactivity. Data are expressed as arbitrary units. *; significantly
different from the control (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
54
Results
Fig. 5.(A) Protein carbonylation. Graphs represent the immunodensity quantitative analysis of three
different immunoblot experiments. (Individual densities from the different bands were added up to
generate one single value). Confluent ECs were stimulated for 80 min as indicated in figure. In
selected experiment, before NA treatment, ECs were preincubated for 30 min with 5mM of the
ROS scavenger NAC. (B and C) Confluent ECs were stimulated for 4 h as indicated in figure.
Hoechst/PI ratio (B) and apoptosis quantification (C) were assessed after treatments as reported in
the „„Materials and Methods‟‟ section. In selected experiment, before NA treatment, ECs were
preincubated for 30 min with 5mM of the ROS scavenger NAC. CTRL, untreated cells; R,
resveratrol; C, coumaric acid; and Stauro, staurosporine. (A and B) Data are expressed as percent
of control.*Significantly different from the control, §significantly different from each other,
#significantly different from each other, and •significantly different from all values (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
55
Results
§ 4.3 Flavin-containing oxidases mediate NA-induced intracellular ROS
production and protein carbonylation
Since flavin oxidases are an important source of intracellular ROS
[112], we planned to investigate their potential involvement in the NAinduced pro-oxidant effect. To this end, we pretreated cells cultures with the
broad flavoproteins inhibitor, diphenylene iodonium (DPI) and then assessed
intracellular ROS generation and protein carbonyl levels in response to NA
treatment. As reported in figures 6A and 6B, DPI significantly blunted ROS
production and protein carbonylation in both resveratrol- and coumaric acidtreated cells, clearly indicating the involvement of DPI-sensitive flavincontaining systems in the observed NA-induced pro-oxidant effect.
Given that ROS generation in NA-treated cells was inhibited by DPI, we
hypothesized that these ROS were being generated, at least in part, by
increased activity of flavin-containing oxidases. Since DPI is a general
flavoproteins inhibitor, we first sought to demonstrate that NA activated DPIinhibitable oxidases activity at the same time of ROS measurement.
Using an established assay [92; 102], we measured the rate of NADH
and NADPH consumption in ECs as an index of cellular oxidase activity in
response to NA treatment. As shown in figure 6C, the rate of NADPH
consumption in NA-treated cells was about 2-fold that of control cells, while
there was no significant change in the rate of NADH consumption.
Subsequently, the effect of DPI on NADH and NADPH consumption was
examined. As observed for NA-induced ROS generation (fig. 6A), DPI was
able to prevent NA-induced NADPH utilization (fig. 6C), clearly implicating
DPI-sensitive flavin oxidases in the cellular response to moderately high NA
concentration. DPI-inhibitable NADPH consumption was also noted in NAunstimulated cells; however, this effect was about one-third of the increase in
NA treated cells. DPI-sensible NADH consumption was observed in control
cells but not in NA-treated cells.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
56
Results
Fig.6. Confluent ECs were stimulated for 80 min as indicated in figure. In selected experiment,
before NA treatment, ECs were preincubated for 15 min with DPI (10µM). (A) Intracellular ROS
levels and (B) protein carbonylation. Graphs represent the immunodensity quantitative analysis of
three different immunoblot experiments. (Individual densities from the different bands were added
up to generate one single value.) (C) NADH and NADPH oxidation. CTRL, untreated cells; R,
resveratrol; C, coumaric acid; and DPI, diphenyleneiodonium. (A and B) Data are expressed as
percent of control. (A–C) *significantly different from the control, §significantly different from
each other, and #significantly different from each other (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
57
Results
§ 4.4 NA dose dependently downregulate Akt phosphorylation
To further investigate the molecular mechanism underlying the effects
of the antioxidants, we tried to identify the signal transduction pathway
responsible for cellular responses previously observed.
For this purpose we studied the protein kinase Akt, the MAPK p42/44MAPK
and SAPK/JNKMAPK because their function may be influenced by ROS and
they are important regulators of death and survival in different cell types,
including endothelial cells [80; 95].
We therefore investigated whether NA, at doses able to affect ROS generation
and cell function, could differentially modulate MAPKs and Akt activation.
The cultured cells were stimulated for 15, 30, 60, and 80 minutes with
different concentrations of NA and processed for Western blotting as
described in Materials and Methods.
Immunoblot analysis from dose-response experiments revealed that Akt
activation mirrored NA-induced antioxidant and pro-oxidant effects. Indeed, a
dose-dependent decrease of Akt phosphorylation was observed at 10 and
25µM (fig. 7), an effect consistent with the observed dose-related pro-oxidant
damage (Figs. 1 and 3). The time-course analysis shows that NA-induced Akt
dephosphorylation started at 15 min and remained persistently evident until
80 min.
On the other hand, the lower dose of NA (0.5µM) elicited a transient
increase of both p42/44MAPK and Akt activation (fig. 7), which is
compatible with the decrease pro-oxidant effect and the improvement of cell
function observed at the same NA concentration (figs. 1 and 3).
Protein expression levels for p42/44MAPK and Akt were unchanged at all
time points tested. Additionally, no changes in both the levels of protein
expression and phosphorylation were observed for SAPK/JNKMAPK in
response to NA treatment. Data presented in figure 8 show no change in both
expression and phosphorylation of p42/44MAPK and SAPK/JNKMAPK in
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
58
Results
response to long-time (2–4 h) treatment with the highest dose of NA.
Interestingly, while Akt protein levels remained constant over the time course,
a significant decrease in Akt phosphorylation was evident at 2 and 3 h after
NA treatment, returning to control level at 4 h (fig. 8).
Fig. 7. Confluent ECs were stimulated as indicated in figure and then processed for
immunoblotting as described in the „„Materials and Methods‟‟ section. (A) Representative
immunoblot of total and phospho-Akt (Ser473), total and phosphop42/ 44MAPK (Thr202/Tyr204),
and total and phospho-SAPK/JNKMAPK (Thr183/Tyr185) for ECs treated with resveratrol. (B)
Graphs represent the immunodensity quantitative analysis of three different immunoblot
experiments using Resveratrol. (C) Graphs represent the immunodensity quantitative analysis of
three different immunoblot experiments using Coumaric acid. (Individual densities from the
different bands were added up to generate one single value.) Data are expressed as arbitrary units.
R; resveratrol. C; coumaric acid. Ratios of individual densitometric results were normalized to actin immunoreactivity. Data are expressed as arbitrary units. (B and C) *significantly different
from its own control, #significantly different from the control and significantly different from each
other within the same time point, §significantly different from the same concentration within all the
experimental time points, and ◦significantly different from the same concentration at the 15-min
time point (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
59
Results
Fig. 8.Confluent ECs were stimulated for the indicated time points (in hours) with 25 µM NA and
then processed for immunoblotting as described in material and methods. A: Representative
immunoblot of total and phospho-p42/44MAPK (Thr202/Tyr204), total and phospho-Akt (Ser473)
and total and phospho-SAPK/JNKMAPK (Thr183/Tyr185) for ECs treated with 25 µM
Resveratrol. B: Graphs represent the immunodensity quantitative analysis of three different
immunoblot experiments using Resveratrol (25 µM). C: Graphs represent the immunodensity
quantitative analysis of three different immunoblot experiments using Coumaric acid (25 µM).
Numbers on the X-axis represent: (1) total and (2) phospho-p42/44MAPK (Thr202/Tyr204), (3)
total and (4) phospho-Akt (Ser473), (5) total and (6) phospho-SAPK/JNKMAPK (Thr183/Tyr185).
(individual densities from the different bands were added up to generate one single value). CTRL;
untreated cells. R; resveratrol. C; coumaric acid. Ratios of individual densitometric results were
normalized to -actin immunoreactivity. Data are expressed as arbitrary units. (B-C) *;
significantly different from its own control (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
60
Results
§ 4.5 Akt dephosphorylation and EC damage are mediated by flavin
oxidases
We next wanted to understand if inhibition of flavin oxidases could
restore Akt function and prevent NA-induced adverse responses.
To this end, both Akt levels and activation (as determined by the
phosphorylation status) were assessed in antioxidant-treated cells in the
presence or absence of DPI. As shown in figure 9A, DPI pretreatment
preserved Akt phosphorylation, suggesting a pivotal role for flavin oxidases
in the downregulation of prosurvival pathways. The rescue of Akt signals was
accompanied by a similar reduction in cell damage confirming the biological
relevance of this mechanism in our vascular model (figs. 9B and C).
Treatment with the only DPI did not elicit any significant adverse effects,
which rules out the possibility of a general DPI toxicity under the current
experimental conditions (figs. 9B and C).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
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Results
Fig. 9. (A) Confluent ECs were stimulated for 80 min as indicated in figure and then processed for
immunoblotting as described in the „„Materials and Methods‟‟ section. In selected experiment,
before NA treatment, ECs were preincubated for 15 min with the flavoprotein inhibitor DPI
(10µM). The upper and lower part of panel A show, respectively, the Akt and phospho-Akt
immunoreactivity (Ser 473) and the quantitative immunodensity. Immunodensity values (total and
phosphorylated) are represented on different graphs sharing the same y-axis. Ratios of individual
densitometric results were normalized to  -actin immunoreactivity. Data are expressed as arbitrary
units. (B and C) Confluent ECs were stimulated for 4 h as indicated in figure. Hoechst/PI ratio (B)
and apoptosis quantification (C) were assessed after treatments as reported in the „„Materials and
Methods‟‟ section. In selected experiment, before NA treatment, ECs were preincubated for 15 min
with the flavoproteins inhibitor DPI (10µM). CTRL, untreated cells, R, resveratrol, C, coumaric
acid; and DPI, diphenyleneiodonium. Data are expressed as percent of maximum. (A–C)
*significantly different from the control (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
62
Results
§ 4.6 Akt activation rescues ECs from oxidative stress damage
Whereas dephosphorylation of Akt plays a central role in driving NAinduced cellular damage, we assumed that the restoration of its activity would
counteract the NA effects.
To test this hypothesis, we preincubated ECs for 30 min with the Akt
activator insulin [98] and then, after exposure to NA, we assessed Akt levels
and activation. As reported in figures 10A and 10B, treatment of ECs with
10µM insulin significantly increased the levels of Akt phosphorylation as
compared to untreated cells.
Pretreatment with insulin prevented Akt dephosphorylation induced by NA,
confirming that Akt activation could be restored. Insulin has been shown to
stimulate Akt via activation of PI3K, an effect that can be blocked by the
PI3K inhibitors Wortmannin and LY-294002 (Hermann et al., 2000).
Preincubation of ECs with 20nM Wortmannin (figs. 10A and 10B) or 10µM
LY-294002 (fig. 11) abolished the protective effect of insulin on Akt
dephosphorylation demonstrating that Akt signaling could be activated
specifically via a PI3K-mediated mechanism.
We next investigated whether restoring Akt activation could rescue
NA-induced ECs damage. To test this hypothesis, we activated Akt using
insulin and then we assessed apoptosis and cell viability upon NA treatment.
Akt activation was able to abolish completely the apoptotic response and to
rescue cell viability (figs. 12A and 12B). Interestingly, Akt activation could
reduce basal levels of apoptosis in cultured cells, an important validation of
our experimental model. Blocking Akt function by using either the PI3K
inhibitor Wortmannin (Figs. 12A and 12B) completely abrogated the
protective effect, further validating Akt as the mediator of the insulin
protective effect. These findings clearly indicate Akt as a crucial mechanism
of cell survival in response to NA-induced oxidative stress.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
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63
Results
Fig. 10. (A and B) Confluent ECs were stimulated for 80 min as indicated in figure and then
processed for immunoblotting as described in the „„Materials and Methods‟‟ section. In selected
experiment, before NA treatment, ECs were pretreated for 30 min with 10µM insulin in either the
absence or the presence of a further 15 min preincubation with the selective PI3K inhibitor
Wortmannin (20nM). The left part of the panel shows the immunoreactivity of total and phosphoAkt (Ser473), while the right part reports the immunodensity quantitative analysis. Immunodensity
values (total and phosphorylated) are represented on different graphs sharing the same y-axis.
Ratios of individual densitometric results were normalized to -actin immunoreactivity. Data are
expressed as arbitrary units. CTRL, untreated cells; R, resveratrol; C, coumaric acid; I, insulin; W,
Wortmannin. Data are expressed as percent of control. (A and B) *significantly different from the
control (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
64
Results
Fig. 11. Confluent ECs were stimulated for 80 minutes as indicated in figure and then processed for
immunoblotting as described in the „„Materials and Methods‟‟ section. In selected experiment,
before NA treatment, ECs were pre-treated for 30 minutes with 10 µM insulin in either the absence
or presence of a further 15 minutes pre-incubation with the selective PI3K inhibitors LY-294002
(10 µM). The left part of the panel shows the immunoreactivity of total and phospho-Akt (Ser473),
while the right part reports the immunodensity quantitative analysis. Immunodensity values (total
and phosphorylated) are represented on different graphs sharing the same y-axis. Ratios of
individual densitometric results were normalized to -actin immunoreactivity. Data are expressed
as arbitrary units. CTRL; untreated cells, R; resveratrol, C; coumaric acid, I; insulin, LY; LY294002. Data are expressed as percent of control. (A-B) *; significantly different from the control
(p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
65
Results
Fig. 12. Confluent ECs were stimulated for 4 h as indicated in figure. Cell viability and apoptosis
were assessed after treatments as reported in the „„Materials and Methods‟‟ section. In selected
experiment, before NA treatment, ECs were pretreated for 30 min with 10µM insulin in either the
absence or the presence of a further 15 min preincubation with the selective PI3K inhibitor
Wortmannin (20nM).
(A) Quantification of Hoechst/PI ratio and (B) apoptosis in cultured ECs in the absence (CTRL) or
presence of the indicated treatments. CTRL, untreated cells; R, resveratrol; C, coumaric acid; I,
insulin; W, Wortmannin; and Stauro, staurosporine. Data are expressed as percent of control. (A
and B) *significantly different from the control, #significantly different from each other,
§significantly different from each other, □significantly different from each other, ◦significantly
different from each other, and •significantly different from all values (p < 0.05).
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
66
Results
§ 4.7 CYP2C9 mediate NA-induced ECs damage
Although liver is the most critical tissue in drug metabolism, CYP 2C9,
an isoform of cytochrome P450, is also constitutively present in the
endothelium and is reported to be a significant source of ROS in coronary
arteries [113; 114].
We then investigated whether the CYP2C9 was involved in the induction of
cell death triggered by natural antioxidants.
To this end, we preincubated ECs for 30 min with a specific inhibitor of
CYP2C9, sulfaphenazole (SPZ) (6 µM) and then, after exposure to NA, we
evaluated apoptosis using the fluorimetric kit APOPercentage (Biocolor Ltd,
Carrickfergus, UK). As shown in fig. 13A, SPZ pre-treated cells didn‟t show
apoptosis in contrast to no pre-treated cells, indicating that the SPZ
neutralizes NA-toxic effects.
The loss of mitochondrial membrane potential (ΔΨm) is a hallmark for
apoptosis death and mitochondrial function. It is an early event preceding
phosphatidylserine externalization and coinciding with caspase activation. For
this reason, we studied also ΔΨm variation by evaluating the changes in
fluorescence intensity of cells, stained with lipophilic cationic dye 5,5‟,6,6‟tetrachloro-1,1‟,3,3‟-tetraethylbenzimidazolylcarbocyanine iodide (JC-1). JC1 has advantages over other cationic dyes because it can selectively enter into
mitochondria and reversibly change colour from green to red as the
membrane potential increases. In healthy cells, with high mitochondrial ΔΨm,
JC-1 spontaneously forms complexes known as J-aggregates with intense red
fluorescence. On the other hand, in apoptotic or unhealthy cells, with low
ΔΨm, JC-1 remains in the monomeric form, which shows only green
fluorescence.
After NA treatment, ECs were incubated with JC1 and analyzed at the
fluorometer.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
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Results
As can be seen from fig. 13B, that shows the relationship between red and
green fluorescence emitted, pretreatment with SPZ annuls the NA toxic effect
on mitochondria, which instead can be observed in cells subjected to
treatment with only NA. The decreasing of mitochondrial membrane potential
suggests that NA-induced oxidative stress may cause mitochondrial damage
and dysfunction in ECs; furthermore this data suggest a role for Cytochrome
P450 (CYP) 2C9 in NA-induced toxicity.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
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Results
Fig. 13. Confluent ECs were stimulated for 4 h as indicated in figure. Apoptosis and MMP assay
were assessed after treatments as reported in the „„Materials and Methods‟‟ section.
In selected experiment, before NA treatment, ECs were pre-treated for 15 minutes with 5 µM of the
CYP2C9 inhibitor sulfaphenazole. (A) Apoptosis and (B) Red/Green JC-1 ratios in cultured ECs in
the absence (CTRL) or presence of the indicated treatments..
CTRL: untreated cells; R: resveratrol; C: coumaric acid; SPZ: sulfaphenazole; and Stauro,
staurosporine . Data are expressed as arbitrary units. * significantly different from CTRL.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
69
Results
§ 4.8 SPZ prevents NA-induced p-Akt down-regulation.
Then we evaluated if SPZ also prevented NA-induced p-Akt downregulation.
As shown in fig 14 SPZ pretreatment preserved Akt phosphorylation,
suggesting that this one could work downstream of CYP2C9 in mediating
cellular responses to NA.
Fig. 14. Confluent ECs were stimulated for 80 min as indicated in figure and then processed for
immunoblotting as described in the „„Materials and Methods‟‟ section. In selected experiment,
before NA treatment, ECs were pre-treated for 15 minutes with 5 µM of the CYP2C9 inhibitor
sulfaphenazole. The upper part of the figure shows the immunoreactivity of total and phospho-Akt,
while the lower part reports the immunodensity quantitative analysis. CTRL: untreated cells; R:
resveratrol; C: coumaric acid; SPZ: sulfaphenazole. Ratios of individual densitometric results were
normalized to -actin immunoreactivity. Data are expressed as arbitrary units. * significantly
different from CTRL.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
70
Results
§ 4.9 Cyclosporine A prevents NA-induced ECs impairment.
Finally we investigated whether the mitochondria were involved in
modulating NA-induced cellular impairment. This is why we treated ECs with
the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporine
A (CsA) and evaluated apoptosis using the fluorimetric kit APOPercentage
(Biocolor Ltd, Carrickfergus, UK).
In fact CsA affects mitochondria by preventing the mitochondrial
permeability transition pore from opening, thus inhibiting cytochrome c
release.
Ciclosporin is believed to elicit its effects by directly binding to the
cyclophilin D protein (CypD) that constitutes part of the mitochondrial
permeability transition pore (MPTP) [115; 116], and by inhibiting the
calcineurin phosphatase pathway.[117; 118].
As shown in fig 15 CSA pre-treatment completely prevented oxidative cell
damage strongly indicating mitochondrial involvement in NA-induced ECs
impairment.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
71
Results
Fig. 15. Quantification of Apoptosis in cultured ECs in the absence (CTRL) or presence of the
indicated treatments. In selected experiment, before NA treatment, ECs were pre-treated for 30
minutes with 2 µM Cyclosporine A a specific MPTP inhibitor. CTRL: untreated cells; R
resveratrol; C: coumaric acid; CsA: cyclosporine A; and Stauro, staurosporine. Data are expressed
as percent of maximum. * significantly different from CTRL.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
72
Discussion
CHAPTER 5
DISCUSSION
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
73
Discussion
The change of intracellular levels of ROS and the consequent activation
of specific signaling pathways induce a coordinated set of integrated
physiological responses in cardiovascular tissue. These include growth of
smooth muscle cells, induction of inflammatory response, impairment of
endothelium-dependent relaxation, and cardiac hypertrophy. Each of these
responses, when uncontrolled, contributes to vascular diseases.
The potential value of antioxidants in treating conditions associated with
oxidative stress is well known to scientists and clinicians and it is of immense
interest to patients.
Oxidative stress is a term used to describe an imbalance between the
production and destruction of reactive oxygen species (ROS), thereby leading
to cellular and tissue injury. The basic properties of oxygen are responsible
for the destructive power of free radicals, in particular, their high reactivity.
Humans consume ~ 250 g of oxygen every day, and of this ~ 3–5% is
converted to O2−• and other reactive species [119]. The damage inflicted by
ROS on cellular and extracellular targets such as membrane lipids, proteins,
and DNA clearly contributes to tissue and organ dysfunction in many
pathological states.
The implication of oxidative stress in the etiology of several chronic and acute
degenerative disorders suggests that antioxidant therapy represents a
promising avenue for treatment. Strategies for the intervention and prevention
of cardiovascular disease require an understanding of the basic molecular
mechanism(s) by prophylactic agents (synthetic antioxidants, dietary
antioxidant factors from food plants and medicinal plants) that may
potentially prevent or reverse the promotion or progression of the disease.
Administration of exogenous antioxidants has been extensively investigated
as a means to attenuate myocardial ischemia-reperfusion injury and to treat or
prevent chronic cardiovascular diseases. Investigations in a variety of animal
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
74
Discussion
models have shown beneficial effects of several drugs. However, clinical
trials have furnished inconsistent results.
Given the central role that the endothelium plays in cardiovascular
homeostasis and the involvement of EC dysfunction in CVD pathogenesis
[92], the EC represent an excellent model to investigate the impact of NA on
vascular (patho)physiology.
Polyphenols are the most abundant antioxidants in the diet. Their total dietary
intake could be as high as 1 g/d, which is much higher than that of all other
classes of phytochemicals and known dietary antioxidants. For perspective,
this is ~ 10 times higher than the intake of vitamin C and 100 times higher
that the intakes of vitamin E and carotenoids [31]. Their main dietary sources
are fruits and plant-derived beverages such as fruit juices, tea, coffee, and red
wine. Vegetables, cereals, chocolate, and dry legumes also contribute to the
total polyphenol intake.
Our results show that for NA such as the polyphenols coumaric acid and
resveratrol, the concentration will determine the overall effects on EC (Figs.
3A–C). While low doses of NA showed an antioxidant effect, surprisingly, a
modest increase in concentration induced a completely opposite result (Figs.
1B and 1C), resulting in cell damage (Figs. 5A–C). A similar behavior has
been reported with other NA in vitro and in vivo experimental models [97;
120], suggesting a dual role for NA in regulating EC biology.
A significant number of reports exist in the literature indicating that
resveratrol can function both as pro-apoptotic and anti-apoptotic agents.
Careful review of the studies on cancer prevention with resveratrol
reveals
that
in
each
case,
resveratrol
was
used
at
high
concentration/dose [10-40 mM] [121 - 125]. In contrast resveratrol
protects hearts in a relatively low dose [5-20 μM] [126 - 129]. This would
tend to indicate that resveratrol provides diverse health benefits in a
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
75
Discussion
dose response manner. There are quite a few studies to describe the dose
dependency of resveratrol towards health benefit.
However, since antioxidant effects can vary depending on cell types and
molecules used, the mechanisms involved in antioxidant-mediated cellular
responses are still largely unknown
Multiple lines of evidence presented in this work implicate flavin
oxidases as key players in the response to high levels of NA. First, the
flavoproteins inhibitor DPI completely blunted the increased oxidases activity
elicited by NA (Fig. 6C), while DPI-sensitive oxidase activity in control cells
was minimal, clearly indicating that NA activated DPI-sensitive flavin
oxidases over the basal level (Fig. 6C). Second, DPI also significantly
counteracted the increased ROS production and protein carbonylation elicited
by NA treatment, thus implicating DPI-sensitive flavin containing systems in
the observed NA-induced pro-oxidant effect (Figs. 6A and 6B). Lastly, flavin
oxidase involvement was further confirmed by DPI‟s ability to rescue the
cellular phenotype induced by NA treatment, preventing apoptosis and cell
death (Figs. 9B and 9C).
Here, we also suggest a role for Akt as a key regulator of EC responses to NA
treatment. Indeed, coumaric acid and resveratrol downregulated Akt
phosphorylation in a dose dependent fashion (Fig. 7), an effect
superimposable with dose response effects observed for ROS generation,
protein carbonylation, and cell function (Figs. 1 and 3). This effect was
mirrored by the increased Akt activation at low NA doses (Fig. 7), which was
consistent with the decreased ROS levels and the improvement of cells
function observed at the same NA concentration (Figs. 1 and 3). In addition,
the ability of DPI to restore NA-induced Akt dephosphorylation and cell
damage indicates that this kinase works downstream of the flavin oxidases,
integrating ROS signals into general cellular responses (Figs. 9A–C). Indeed,
Akt is a central signaling molecule in regulating survival and death of
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
76
Discussion
different types of cells including EC, and its activity can be redox regulated
[95; 80]. Rescue of Akt phosphorylation and recovery of cell function after
insulin pretreatment indicate that this kinase is pivotal in mediating NAinduced effects (Figs. 10 and 12). In fact, its inhibition completely abrogated
cell protection, further validating Akt as the mediator of the insulin protective
effect (Fig. 10). Given the complete lack of insulin-mediated protective
effects of Akt on NA action, Akt-independent signals, potentially activated by
insulin, are unlikely to be involved in the investigated phenomena.
Furthermore in the present study we found that treatment of ECs with the
mitochondrial permeability transition pore (MPTP) inhibitor cyclosporine A
(CsA), completely prevents oxidative cell damage strongly indicating
mitochondrial involvement in NA-induced ECs impairment (Fig.15).
Finally NA-induced pro-oxidant effects were counteracted by sulfaphenazole
(SPZ), suggesting a role for Cytochrome P450 (CYP) 2C9 in NA-induced
toxicity (Fig. 13). SPZ also prevented NA-induced p-Akt down-regulation
and mitochondrial membrane potential (MMP) impairment (Figs 13, 14),
indicating that Akt can work downstream of CYP2C9 in mediating cellular
responses to NA.
Our study is the first to show in a human vascular model that moderately
high-doses of NA can induce mitochondrial-dependent cell damage mediated
by CYP2C9- and the Akt pathway. We believe the present results are of
particular importance in light of the popularity of antioxidant rich diets and
therapeutic approaches aimed at reducing cardiovascular risk.
In fact the toxicology of NA has become a controversial area of debate. Our
results support the idea that high doses of NA rather than producing protective
antioxidant effects may prompt pro-oxidant-induced damage. Confirming
previous observations [109; 110], we also reported that low concentrations of
NA can prompt a significant antioxidant effect. Such a phenomena may be
explained by the interaction of NA with lipid rafts/caveolae, specialized
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
77
Discussion
plasma membrane microdomains involved in ROS compartmentalization, and
redox-regulated signal transduction [162]. Indeed, recent data indicate that
nanomolar concentration of resveratrol can enhance endothelial NO
production through a caveolae-dependent mechanism involving p42/
44MAPK activation [110]. Resveratrol at concentrations attainable with
moderate wine consumption (0.1– 0.5µM) has also been reported to activate
Akt, increase NO production, and inhibit NADPH oxidase-dependent ROS
generation in human platelets [109]. Our results, showing reduced ROS levels
in association with p42/44MAPK and Akt activation in response to low dose
of NA, suggest that a similar mechanism may be conceivable. Indeed in
addition to resveratrol, improved endothelial function via a caveolae-mediated
antioxidant effect has been also reported for other NA, such as quercetin and
red wine polyphenols [131; 132]. Surprisingly, the effects of NA have been
mainly tested against oxidative-induced damage or other toxic insults,
whereas relatively little effort has been put in assessing their potential effects
under normal conditions. For example, resveratrol at a concentration of
50µM, well above the concentrations currently used in this work, protects
HUVEC from oxidized low-density lipoprotein–induced oxidative damage
[133]. However, it has been suggested that resveratrol can work as a prooxidant under low oxidative conditions, while it becomes antioxidant under
strong oxidative conditions [134]. Thus, the interaction of NA with the
cellular redox state could be of primary importance, especially when precise
redox modulation is needed to allow normal cell function or to promote cell
death. In vivo, there is evidence suggesting that NA can accumulate in
specific compartments at relatively high concentrations. For example, after
chronic consumption, resveratrol has been shown to be detectable in plasma
up to 1 week after washout [135], and plasma peak concentrations of 32 and
8.1µM have been reported in rodents [136; 137]. Because of the lipophilic
nature of most NA, their tissue levels may provide a better indicator of the in
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
78
Discussion
vivo biologically active concentrations outlasting their presence in the plasma.
Indeed, in accordance with the reported ability of ECs to uptake NA [138], in
rats fed dietary relevant doses, concentrations of resveratrol in tissues such as
the heart, liver, and kidney were higher (~10 to 30µM) than in plasma [139;
140]. Also, it has been suggested that plasmatic proteins may be natural NA
reservoirs in vivo, modulating their plasma concentration and tissue delivery
[138; 141]. Moreover, interactions between different NA may also influence
their kinetics and metabolism in the liver increasing NA circulating levels
[142]. Interestingly, several studies consistently indicate that resveratrol
metabolites half-life, and concentration in plasma is 10 times higher
compared to that of the native compound [143] and whether these metabolites
may serve as pool from which free resveratrol can be released locally in
various tissues cannot be excluded at the moment. Such an aspect seems to be
relevant also in clinical practice. Although some studies demonstrate a
significant inverse correlation between NA-rich foods consumption and
cardiovascular risk, others indicate that NA fail to protect against CVD or
may accelerate their development [36]. Evidence suggests that the oxidant
and antioxidant status of people should be probably checked before
undergoing high antioxidant intake or supplementation [144; 145]. The
human population is heterogeneous regarding the ROS level. Screening the
human population regarding innate or acquired ROS levels can provide the
necessary information about individual oxidative status. High doses of
antioxidants can reduce the ROS level in people who over produce ROS and
protect them against cancer, cardiovascular diseases and other ROSdependent morbid conditions. For people with a low ROS level, high doses of
antioxidants can be deleterious, suppressing the already low rate of ROS
generation and the ROS-dependent cancer preventive apoptosis. Screening
and monitoring the human population regarding the ROS level can transform
antioxidants into safe and powerful disease-preventive tools.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
79
Discussion
To this regard is worth noting that NA are not only part of the human diet but
are often ingested as an additional dietary supplement at high dosage or used
in clinical trial at pharmacological doses. For example, commercial dietary
supplements contain on average between 50 and 325 mg resveratrol, with
some high-potency varieties containing up to 500 mg. Supplement doses
range from a daily dose of 50 to as high as 2000 mg/day (0.8–33 mg/kg body
weight/day for a 60 kg human). Despite present diffuse contradictions, it
appears that at least populations with insufficient or unbalanced nutritional
levels may benefit from an increased intake of dietary antioxidants or
supplements [146]. Thus antioxidant supplementation could potentially be
harmful to those tissues that are not subjected to substantial oxidative stress.
Conversely, for those disorders that are associated with marked increases in
ROS production the temporal and spatial characteristics of oxidant production
pose great challenges in regard to delivering effective antioxidant therapy.
Instead, the methods currently available to assess the degree of oxidative
stress, and the efficacy of antioxidant therapy, in vivo are quite limited. To
our knowledge, organs and tissue levels of NA in people under
pharmacological treatment or supplementation with high doses of NA are so
far unknown. Ideally, antioxidant therapies should be judged on the basis of
their therapeutic efficacy. Unfortunately, determination of the efficacy of
antioxidant therapy is hampered by the lack of available methodology to
quantify ROS in tissues and blood vessels in vivo. Surrogate end points, such
as assessment of endothelial function or lipid peroxidation products in the
plasma, do not adequately reflect the capacity of antioxidants to protect the
deeper layers of the blood vessel wall from oxidative injury. Negative results
of clinical trials must be interpreted cautiously in the absence of verification
that antioxidant therapy successfully reduces vascular oxidant stress.
Moreover, ROS may participate only in certain subsets of vascular diseases
and/or in specific patient subpopulations. The aforementioned potential
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
80
Discussion
pitfalls of antioxidant therapy might be considered theoretical rather than
pragmatic. Although antioxidants are typically given in constant amounts and
dosing intervals, oxidative stress is not a continuous, uniform process. For
example, marked intensification of oxidative stress occurs transiently after
vascular balloon injury, and, most likely, during periods of increased
inflammatory activity in atherosclerotic lesions [147]. The oxidants may
activate signaling cascades and gene expression that, once set in motion, no
longer require the presence of ROS.
Although further studies are required to better characterize the molecular
mechanism of the NA-induced cell toxicity, our findings support recent
observations suggesting that NA can have a potent nonspecific toxicity
towards normal cells [148]. It remains to be elucidated whether NA-induced
EC toxicity could help to explain some of the mixed results obtained with
NA-based strategies in the prevention or treatment of cardiovascular
pathologies.
Much remains to be learned concerning the signaling pathways and genes that
are regulated by ROS. Because redox-sensitive responses appear at times to
be cell specific, it will be important to identify the sources of oxidant stress in
each cell, the mechanism of regulation of antioxidant enzymes and the effect
of ROS on signaling pathways specific to the function of that particular cell
and to gain further insight into the physiological responses affected by
oxidant stress. An understanding of these events will enable us to devise
therapeutic strategies to target specific cellular events contributing to vascular
disease. It would also be necessary to establish optimal doses of antioxidants
capable of coping with high and low levels of ROS.
However, since there is a substantial body of published work that shows NA
can reach in vivo concentrations comparable to the ones we used in vitro, we
suggest that our results could be representative of a physiologically relevant
in vivo mechanism.
Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
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Dott.ssa Annalisa Cossu
“Study of intracellular signaling pathways triggered by natural antioxidants in human endothelial cells”
Tesi di Dottorato in Scienze Biomolecolari e Biotecnologiche – Università degli studi di Sassari
91